Application of antibiotics of the phleomycin family as selection agent in the field of genetic engineering

ABSTRACT

A method of transforming eukaryotic or prokaryotic hosts sensitive to an antibiotic of the phleomycin family to confer resistance to the antibiotic is disclosed in which a phleomycin resistance gene is used as a selectable marker.

The present invention relates to the application of phleomycins as aselection agent in the field of genetic engineering.

For bacteria there are available at the present time reliable andsensitive selection markers which are constituted by characters ofresistance to certain antibiotics such as ampicillin, on the other hand,for eucaryotic cells the selection labelers are, generally, insensitiveor require the use of a complicated technology to be usable.

It is, in addition, interesting to have available a selection labelerwhich can be expressed both in prokaryotic cells and in eukaryotic cellssince often the vector constructions in the eukaryotic cells are partlyeffected in prokaryotic cells.

The present invention rests on the demonstration of new selectionlabelers which confer on the cells a resistance to antibiotics of thephleomycin type.

The present invention relates to the application of antibiotics of thephleomycin family as selection agent of cells which have beenartificially modified by incorporation of a phleo^(r) gene of resistanceto an antibiotic of the family of phleomycins.

By "antibiotic of the family of phleomycins" is meant to denote, besidesthe phleomycins themselves, related antibiotics such as bleomycins,zorbamycins, victomycin, platomycins, tallysomycins or the antibioticsSF 1771, SF 1961 and YA 56, as well as mixtures of these antibiotics.

These antibiotics are characterized by a basic structure whichdifferentiates them distinctly from the other antibiotics and the testscarried out within the scope of the present invention have demonstratedthat the exact nature of the antibiotic used for the selection does notnotably modify the result obtained provided that the antibiotic isindeed of the phleomycin type.

These antibiotics obtained commercially (bleomycine) or prepared fromproductive strains ATCC 21890 and 21892 (phleomycins), ATCC 21807(victomycine), ATCC 21893 (platomycines), ATCC 31158 (tallysomycines),ATCC 31248 (SF 1771) are relatively toxic for bacteria, the lowereukaryotics like the yeasts and the fungi and the higher eukaryoticsboth animal and plant cells in cultivation.

Recently isolated strains of Actinomycetes produce antibiotics identicalwith those mentioned above or different. Particularly, the strain V9,Streptoverticillium in the course of taxonomic identification,synthesizes substances belonging to the family of phleomycines. Theabsorption curves in the ultra-violet of the purified products of the V9strain are significantly different from those of the products mentionedabove.

The genes which confer resistance on antibiotics of the phleomycin typeare named below generically as phleo^(r) genes.

It should be pointed out that up to the present numerous distinctphleo^(r) genes have been established as will be demonstrated below.

The phleo^(r) genes are borne by natural plasmids isolated frombacterial strains, for example found in a hospital environment, or canbe cloned from the DNA of various organisms, particularly the DNA oforganisms producing antibiotics of the phleomycin type. But thesephleo^(r) genes also exist in genetic materials widely available sinceit has been possible to establish the presence of such a gene on thetransposon Tn5 of Escherichia coli or on the plasmid pUB110.

A gene of great importance for the present invention is that borne bythe transposon Tn5 to E. coli well known to molecular biologists. Thisgene whose function has not yet been described is situated in thecentral portion of Tn5, between the gene which results in the resistanceamong others to kanamycin and that which results in the resistance incertain bacteria to streptomycin.

The exact location of the gene concerned, denoted by Tn5 phleo^(r), hasbeen determined exactly by dissection of the Tn5 DNA with variousrestriction enzyme and by determination of the corresponding nucleotidicsequence.

The phleo^(r) Tn5 gene results in the resistance to all the antibioticsmentioned above, including here those of the V9 strain, when the latteris rendered functional after attachment of suitable promoter andterminal sequences of the carrier organism.

Thus the phleo^(r) Tn5 gene alone is functional in Gram⁻ bacteria and inGram ⁺ bacteria like for example the bacilli and the streptomyces.

The phleo^(r) Tn5 gene results also in the resistance to theseantibiotics in yeasts, to animal and vegetable cells in cultivation whenthe latter contain the functional gene.

A second gene worthy of interest is borne by the plasmid pUB110 wellknown to geneticians of the bacilli. This gene is situated on theplasmid pUB110 downstream of the gene which confers the resistance totobramycin. It is comprised in a region bounded by the BglII site andwhich ends in anticlockwise direction to the BamHI site.

This gene named phleo^(r) 110 results in resistance to all theantibiotics mentioned previously in bacilli bearers of the plasmidpUB110 and also in Gram⁻ bacteria when the gene is placed under thedependence of suitable promoter and terminal sequences.

By analogy with the example of the phleo^(r) Tn5 gene the phleo 110 geneshould also be expressed in the environments of lower and highereukaryotes after attachments to the sequences specific to each organism.

A third source of resistance genes to antibiotics of the family ofphleomycins is provided by bacteria producers of these antibiotics.

A gene denoted phleo^(R) SV present on the chromosomic DNA of the SVstrain has been cloned on a streptomyces plasmid. The phleo^(R) SV genesituated on the hybrid plasmid pUT212 confers the resistance to theantibiotics of the family of phleomycin, to the Streptomycesviolaceoniger strain which harbors the plasmid.

The physical maps of the genes Tn5 phleo^(r), 110 phleo^(r) and SVphleo^(r) determined by various restriction enzymes are dissimilar,which indicates a very great divergence between the three genes.

The employment of the antibiotics of the family of phleomycins, whichwill sometimes be called below phleomycins for simplification, asselection agent may be done according to modalities known in the fieldof genetic engineering.

Thus, when the technique employs autoreplicating vectors such asplasmids, phages or viruses, the selection agent is generally usedpositively or negatively.

It is possible thus to amount to a process of selection of a transformedor transfected cell by a cloning and/or expression vector of adetermined protein, characterized in that there is introduced into thisvector a phleo^(r) gene of resistance to an antibiotic of the family ofphleomycins as well as the elements possibly necessary to ensure itsexpression into the cells concerned, and in that, after transformationor transfection, cells obtained are subjected to the action of anantibiotic of the family of phleomycins and in that the resistance cellsare selected.

It may also amount to a process of selection of transformed ortransfected cells by a cloning and/or expression vector of a particularprotein which has integrated a DNA segment, characterized in that a DNAsegment is introduced into the phleo^(r) gene of resistance to theantibiotic of the family of phleomycins borne by said vector and thetransformed or transfected cells are subjected to the action of anantibiotic of the family of phleomycins and the sensitive cells areselected.

This second process obviously necessitates having preserved an originalof the culture to be able to take up therefrom, the sensitive colonies.

These processes are well known in principle, however when, the cellsemployed are eucaryotic cells, the yields of selection with labelers ofthe prior art are often rather low and necessitate the use of largeamounts of the selection agent, which in certain cases only ensures avery low selection margin.

On the contrary, the use of the phleomycins permits a selection both inprokaryotes and in eukaryotes with small amounts of selection agent andwith very high sensitivity.

By way of example, in the case of the yeast Saccharomyces cerevisiae,the strain transformed by a vector bearing the phleo^(r) gene resists toconsiderable concentrations of bleomycins, 100 times greater than thosesupported by the untransformed strain.

Generally, the selection agent will use concentrations comprised between0.1 and 100 γ in the culture medium of the modified cells.

Thus, it has been possible to establish the action of the selectionagents, both in Gram⁻ and Gram⁺ bacteria, and in yeasts, fungi or animalor vegetable cells.

In addition, although it is interesting to insert the phleo^(r) gene ina vector, it is also possible to integrate this gene into a cellularchromosome in order to confer on the cell a resistance to thephleomycins.

Of course, the structure of the previously mentioned vectors does notconstitute in itself a characteristic of the present invention and thesevectors may be prepared by known techniques. It is besides evident thata certain number of vectors existing at present could be adapted byinserting the phleo^(r) gene so as to be able to be manipulated by usingthe selection agents of the present invention.

The elements necessary to ensure the expression of the phleo^(r) genedepend, of course, on the host cell, but are known to the man skilled inthe art; the examples below will demonstrate besides certain elementsenabling the expression of phleo^(r) gene to be ensured according to thenature of the host.

These examples will be described by referring to the figures in which:

FIG. 1 is a diagram of the plasmid pUT3,

FIG. 2 shows a nucleotidic sequence of the phleo^(r) gene of thetransposon Tn5,

FIG. 3 is a diagram of the plasmid pUT100,

FIG. 4 is a diagram of the plasmid pUT201,

FIG. 5 is a diagram of the plasmid pUT6,

FIG. 6 is a diagram of the plasmid pUT66,

FIG. 7 is a diagram of the plasmid pUT8,

FIG. 8 is a diagram of the plasmid pUT301,

FIG. 9 is a diagram of the plasmid 400,

FIG. 10 shows diagramatically the restriction fragments of pKC7 employedin the examples.

The abbreviations used in these figures are considered as known or willbe explained in the description.

Except for indication to the contrary, the various processes andproducts mentioned are employed according to techniques which are knownand/or recommended by the manufacturer.

EXAMPLE I

Cultures in a Petri dish are prepared from Actinomycetes strainsproducing antibiotics belonging to the group of Bleomycins, obtainedfrom the American Type

    ______________________________________                                        Culture Collection:                                                           ______________________________________                                        Streptomyces verticillus                                                                        ATCC 15003 (Bleomycins)                                     Streptomyces verticillus                                                                        ATCC 21678 (Bleomycins)                                     Streptomyces verticillus                                                                        ATCC 21890 (Phleomycins)                                    Streptomyces flavoridis                                                                         ATCC 21892 (Phleomycins)                                    Stretoalloteichus hindustanus                                                                   ATCC 31158 (Tallysomycins)                                  Streptosporangium violaceo-                                                                     ATCC 21893 (Platomycins)                                    chromogenes sub. sp. globophilum                                              Streptosporangium ATCC 21807 (Victomycins)                                    violaceochromogenes                                                           Streptomyces toyocaensis                                                                        ATCC 31248 (S.F. 1771)                                      ______________________________________                                    

The surface of the dishes containing 30 ml of GAPY (glucose 10; solublestarch 20; yeast extract 5; soja peptone 5; CaCo₃ 1; gelose 15 g/l per 1l of distilled water) is seeded by a platinum loop with a suspension ofmycelia spores of the above strains. The production of the antibioticsis estimated after 15 days of incubation at 27° C. in the followingmanner: a gelose cylinder is taken up from the dish of the productionmedium then deposited on a medium (antibiotic n°2 DIFCO+2 g/l glucose,pH 8) seeded in depth with bacteria of the strain HB101 of Escherichiacoli. In this example two indicating strains are used: HB101 and HB101containing the plasmid pKC7 (RAO et al., Gene 7, 79, 1979). The plasmidpKC7 isolated from the strain ATCC 37084 was introduced by atransformation in the strain HB101.

The results of the antibiogram expressed in inhibition diameter (mm) areshown in table 1.

                                      TABLE 1                                     __________________________________________________________________________    Determination of the sensitivity of two isogenic strains of                   Escherichia coli to the antibiotics of the family of bleomycins               Indicating                                                                          Producing Strains                                                       Strains                                                                             15003                                                                             21678                                                                             21890                                                                             21892                                                                             31158                                                                             21893                                                                             21807                                                                             31248                                       __________________________________________________________________________    HB 101                                                                              31  32  17  18  37  28  16  14                                          HB 101                                                                               6  --  --  --  20  --  --  --                                          (pKC7)                                                                        __________________________________________________________________________

It emerges from this table that the plasmid pKC7 protects the cellswhich harbor it against all the antibiotics of the family of bleomycinsexamined in this example. The strain 31158 producing tallysomycins alsosynthesizes antibiotics of the aminoside type among which areparticularly apramycin. The apramycin tested by the method of discs (50μg) gives an inhibition aureola of 30 mm identical for the twoEscherichia coli strains. The inhibition observed for the strain HB101(pKC7) by the gelose cylinders of the strain 31158 is due to thepresence of the apramycin.

EXAMPLE II

The gelose medium corresponding to 20 Petri dishes incubated for 10days, of production medium of the strain Streptoalloteichus hindustanus(ATCC 31158) is ground twice by means of a ultraturax in the presenceeach time of 200 ml of water then centrifuged at 5000 rpm. Thesupernatant liquor is filtered, then passed into a column of resinIRC-50 previously equilibrated with trifluoroacetic acid (0.1N). Thetallysomycins present in the supernatant liquor are absorbed on theresin whereas all the aminosides (nebramycins, tobramycin and apramycin)are not retained. After washing with water, the tallysomycins aredetached with 0.01N trifluoroacetic acid. The volume of the eluate isreduced to half by evaporation under reduced pressure and the pH broughtto 7. The resulting solution kept at 4° C. is the solution oftallysomycins used in the remainder of the tests. The cellulose discinbibed with 20 μl of this solution gives an inhibition aureola of 33with HB101 and no inhibition with HB101 (pKC7) under the conditionsdescribed in example I.

A bleomycin solution constituted principally of the component A5 isprepared in identical fashion from 20 dishes of the Streptomycesverticillus (ATCC 21678) strained supplemented for this strain with 100μg/ml spermidine. A cellulose disc soaked with 20 μl of this A5bleomycin solution gives an inhibition aureola of 35 with HB101 and doesnot give an inhibition with HB101 (pKC7).

EXAMPLE III

The productivity of the strain ATCC 21890 of Streptomyces verticillusbeing low in stirred liquid cultures, a genetic improvement of thestrain was effected by two mutagenic treatments and selection by themethod of the gelose cylinder.

    ______________________________________                                        Strain 21890→A 152-1→B-81 C                                     ultra-violet light                                                                           nitroso-guanidine                                              ______________________________________                                    

The mutant strain B-81 C was used for a production of phleomycins inliquid culture: an Erlenmeyer flask of 300 ml containing 40 ml of theinoculum medium (soluble starch: 10; yeast extract: 5; acid caseinhydrolysate: 5; meat extract: 3; glucose: 1; MgSO₄ : 0.5; KH₂ PO₄ : 0.2;NaHPO₄ : 0.2; CaCO₃ : 0.5; g per liter, pH 7 before sterilization) isseeded with 0.5 ml of a suspension of mycelium-spores prepared from adish of GAPY medium of the mutant strain B-81 C of Streptomycesverticillus. After 40 hours of incubation at 27° C. on the rotarystirrer (220 rpm) 5 ml of the inoculum culture were used to inoculate anErlenmeyer flask of 2 liters containing 200 ml of the production medium(soluble starch: 20; glucose: 10; Pharmamedia: 10; soluble corn extract:10; ammonia sulfate: 3; CaCO₃ : 4 g per liter, pH 6.5 beforesterilization). After 7 days of fermentation at 27° C. on a rotarystirrer (250 rpm/min) the culture corresponding to 5 Erlenmeyer flasksis filtered. The filtrate (900 ml) is passed into a colony (40 cm×2 cm)of IRC-50 resin previously equilibrated with trifluoroacetic acid(0.1N). After washing with 1 liter of distilled water the phleomycinsare eluted with 300 ml of 0.02N trifluoroacetic acid. The neutralizedeluate is passed directly over a column (30×2 cm) of XAD-2 resinpreviously equilibrated with water. After washing the column with 250 mlof distilled water, the phleomycins are eluted with 100 ml of 80%methanol in 0.1N HCl. The eluate evaporated to dryness gives 210 mg of ablue powder constituted principally of phleomycins complexed with Cu⁺⁺.The powder obtained by this procedure is free from antibiotic active onGram⁺ bacteria distinct from bleomycins and from the polyenic antifungicagent produced conjointly by the strain 21890 and its mutants. Theactivity of the phleomycin powder is compared with that of bleomycinmarketed by the Laboratoires Roger Bellon (France) and the pepleomycinunder clinical testing (Laboratoires Roger Bellon). The table 2indicates the results of the antibiogram of discs charged with 20 μl ofsolutions with 1 mg/ml of each of the antibiotics. The solutions of bluecolor of bleomycin and of pepleomycin complexed with Cu⁺⁺ are preparedby addition of cupric chloride to the white solutions of bleomycin andpepleomycin. The tetracycline is employed as a control at the sameconcentration. In this example two strains of Bacillus subtilis areadded. The strain 168 is well known to geneticians of bacilli, in thesame way as the plasmid pUB110 which was introduced by transformationinto the strain 168.

                                      TABLE 2                                     __________________________________________________________________________    Antibiogram of two isogenic strains of Escherichia coli and of two            isogenic stains of Bacillus subtillis with antibiotics of the bleomycin       family                                                                        (antibiotic medium DIFCO n° 2 + 2 g/l glucose, pH 8, discs of 10       mm)                                                                           Indicator                                                                           Phleomycin  Bleomycin    Pepleomycin                                    strains                                                                             Cu.sup.++                                                                           Bleomycin                                                                           Cu.sup.++                                                                           Pepleomycin                                                                          Cu.sup.++                                                                            Tetracycline                            __________________________________________________________________________    Escherichia                                                                   coli                                                                          HB 101                                                                              31    25    24    26     27     21                                      HB 101                                                                              --    --    --    --     --     22                                      (PKC 7)                                                                       Bacillus                                                                      subtillis                                                                     168   34    26    26    30     29     28                                      168   --    --    --    --     --     28                                      (pUB 110)                                                                     __________________________________________________________________________

It emerges very clearly from these results that the plasmid pKC7 on theone hand and the plasmid pUB110 on the other hand protect their hostcells against the toxic effect of the different components of thephleomycins and of the bleomycins. The nature of the terminal amine ofthe bleomycin nucleus or of the phleomycin nucleus as well as the formcomplexed or not with the Cu⁺⁺ do not have an influence on thisprotection.

EXAMPLE IV

The plasmid pKC7 which results in the resistance to kanamycin and tobleomycins due to the fact of the fragment HindIII-BamHI of thetransposon Tn5 is deleted from the small fragment SalI-SalI aftercomplete digestion and ligation. The cells HB101 containing theresulting plasmid pUT2 are resistant to kanamycin but sensitive tobleomycins. The plasmid pUT3 (FIG. 1) which results from the ligation ofthe large fragment HindIII-SalI of pUT2 (digestion of pUT2 by SalI andHindIII and electroelution from agarose gel) and of the small fragmentHindIII-XhoI of pKC7 (cleavage of pKC7 by HindIII and XhoI thenelectroelution) results in resistance to kanamycin and to bleomycins.

The transformation of HB101 by the DNA of the plasmid pUT3 cleaved withSpHI and then digested with Ba131 for variable times (5 min., 10 min. 20min.) followed by addition of phosphorylated BglII linkers and BglIIcleavage and then ligation gave resistant ampicillin transforming clonesall sensitive to kanamycin and for about half resistant to bleomycins at5 min times (at 10 and 20 min times all the clones were sensitive tobleomycins). The plasmid pUT37 is extracted from one of the clonessensitive to kanamycin and resistant to a high concentration ofcommercial bleomycin.

These results show very clearly that the gene of resistance to thebleomycins of the transposon Tn5 is distinct from that which codes for a3' aminoside phosphotransferase of type II responsible for theresistance to kanamycin and geneticine. The sequence of the gene ofstructure named indifferently Tn5 Phleo^(r) or Tn5 Bleo^(r) is given inFIG. 2.

EXAMPLE V

The small fragment BglII-BamHI of pKC7 obtained by double digestion andelectroelution of agarose gel was ligatured with the large fragmentBglII-BamHI of the plasmid pUB110 of Bacillus subtilis. Aftertransformation of the protoplastes of the strain 168 of Bacillussubtilis the clones obtained on the DM3 regeneration medium supplementedwith 100 μg/ml of kanamycin were checked by their sensitivity totobramycin (10 μg/ml, DIFCO antibiotic n°2 medium, pH 8) and theirplasmid extracted. The size of the majority of the plasmids showed theexpected value for the construction desired with the fragment insertedin a single direction, that where the two sites BglII and BamHI arerestored (plasmid pUT100--FIG. 3). The cells of Bacillus subtiliscontaining the plasmid pUT100 are resistant to commercial bleomycin andto the same aminosides as those inactivated by the APH3'II of thetransposon Tn5. Thus the fragment BglII-BamHI of the portion of Tn5borne by pKC7, devoid of promoter region expresses the two types ofresistance described in the structure described for this example, inBacillus subtilis Gram⁺ bacteria.

In the same way, the character of resistance to commercial bleomycin canbe obtained in the Streptomyces, filamentous bacteria of greatimportance in the fermentation industry.

The small fragment BglII-BamHI of pKC7 was inserted at the site BglII ofthe plasmid pIJ702 (KATZ et al., Journal of General Microbiology, 1983,129, 2703). The hybrid plasmid resulting pUT201 (FIG. 4) characterizedby double digestions results in the resistance to kanamycin and tocommercial bleomycin when the latter is present in Streptomyces lividansand Stretomyces violaceoniger.

EXAMPLE VI

This example is given as an illustration of the interest of thebleomycins to select clones transformed in the case of experiments ofgenetic transfer in higher eucaryotic cells.

The small fragment HindIII-SalI of pUT37 is ligatured with the largestfragment HindIII-SalI of pKC7 to give the plasmid pUT6 (FIG. 5). Thisplasmid pUT6 is cleaved with XhoI, the adhesive ends rendered sharp bythe action of Klenow polymerase and after cleavage by BglII, the smallsharp-ended fragment BglII-XhoI is electroeluted from agarose gel. Thefragment is ligatured with the large fragment BglII-SmaI of the plasmidpAG60 (COLBERE-GARAPIN et al., J. Mol. Biol. (1981) 150, 1) to give theplasmid pUT66 (FIG. 6).

The DNA of plasmid pUT66 is used for the transfection of the line L ofdeficient thymidin kinase mouse cells (TK ) by the method described byCOLBERE-GARAPIN et al., Proc. Nat. Acad. Sci. USA (1979), 76, 3755).Addition of 75 μg/ml of commercial bleomycin to the growth mediumresults in a lethality of all the cells in 10 days of incubation.Addition of 100 μg/ml of commercial bleomycin two days after thetransfection (2 μg of DNA of pUT6 per 10⁶ cells by dish) followed by 3weeks incubation, enables the selection of 50 clones on the average perdish transformed for the character of resistance to the bleomycin. Thenumber of transformed clones is essentially the same when the selectionis made and the two concentrations tested: 100 μg and 200 μg/ml ofcommercial bleomycin. It is important to indicate that when the plasmidresulting from the replacement of the fragment BglII-XhoI of pUT66 bythe corresponding fragment of pKC7 is used to transfer the mouse cells,only the transformants for the resistance to geneticine (G418) but notto the bleomycins can be selected. This result is in accordance with thefact that in eukaryotes, contrary to prokaryotes cells, the translationof a polycistronic messenger RNA is not reinitiated after a translationtermination codon.

EXAMPLE VII

This example is for the purpose of showing the advantage of bleomycinsfor the selection of transformed clones in lower eukaryotes andparticularly Saccharomyces cerevisiae.

The large fragment EcoRI-AsuII of pKC7 obtained by EcoRI and AsuIIdouble digestion, electroeluted from an agarose gel is treated withKlenow polymerase to transform the sticky ends into clean ends, followedby ligation. The transformation of HB101 gives clones resistant toampicillin, weakly resistant to commercial bleomycin. The minimuminhibiting concentration of these carrier clones of the plasmid pUT8(FIG. 7) is 0.5 μg/ml by comparison with 0.1 μg/ml for the strain HB101and 100 μg/ml for HB101 (pKC7) in the antibiotic medium n°2+2 g ofglucose, pH8. The low resistance to bleomycin resulting from the pUT8 isdue to the complete loss of the promoter sequence (Pribnov box andfixation sIte of the ribosomes) in the deleted region. The EcoRI andAsuII sites are restored in this construction. The DNA of the pEX-2plasmid (GRITZ et DAVIES, Gene (1983) 25:179) is linearized by completedigestion with BamHI. After partial digestion with EcoRI, the fragmentwhose size corresponds to the linear plasmid is electroeluted from anagarose gel and ligatured with the small fragment EcoRI-BamHI of pUT8.After transformation of HB101, one of the clones selected on ampicillinand resistant to 2 μg/ml of commercial bleomycin is retained as sourceof the plasmid pUT301 (FIG. 8).

The strain OL 1 (α, leu 2-3, leu 2-112, his 3-11, his 3-15, ura 3-251,ura 3-373, BOY MARLOTTE et JACOUET, Gene (1982) 20:433) of Saccharomycescerevisiae is transformed by the DNA of pUT301 according to the methoddescribed by KLEBE et al. Gene (1983) 25:333) modified in the followingmanner: the cells treated with polyethylene glycol for 1 hour to 30minutes are washed once and resuspended in 5 ml of 60% YPD-40% NBmedium. After 6 hours of incubation at 30° the cells are centrifuged andthe culot taken up again with 1 ml NB medium. The cells after dilutionare spread with a rake either in the YNB medium supplemented with 100μg/ml of histidine and 100 μg/ml of leucine, or in the medium DIFCOn°2+5 g/l glucose, pH 7.5, supplemented with 50 μg/ml of commercialbleomycine. After 3 days of incubation at 30° C. the number of clonestransformed for the character of resistance to bleomycine issubstantially the same as the number of those transformed for thecharacter ura⁺ (10³ transformed per μg of DNA). Check of the clones hasshown on the one hand that all the ura⁺ clones were resistant to atleast 150 μg/ml of commercial bleomycin and on the other hand the clonesselected for resistance to bleomycin were all ura⁺.

EXAMPLE VIII

This example is intended to show that the gene of resistance tobleomycin of pUB110 is different from that which codes for the synthesisof the aminoglycoside transferase AAD 4' responsible for the resistanceto kanamycin and to tobramycin.

The insertion of Sau3A fragments of the DNA of bacteriophage 4 at thesite BglII of pUB110 gives clones after transformation of theprotoplasts of Bacillus subtilis resistant to bleomycin but sensitive tokanamycin and to tobramycin (plasmids of the series pUT102). Conversely,deletion of the two smallest fragments HpAII of pUB110 (partialdigestion of the DNA of pUB110 with HpaII and electroelution from anagarose gel of a band of about 3.7 kilobases closed back on itself byligation) leads to the plasmid pUT101 which expresses in Bacillussubtilis the resistance to kanamycin and not to bleomycin.

EXAMPLE IX

The preceding examples have shown that two distinct genes of resistanceto the antibiotics of the family of bleomycins are found carried one bythe transposon Tn5 of Escherichia coli and on the other hand by theplasmid pUB110 of Staphylococcus and Bacillus subtilis. The source ofresistance gene to the bleomycins is not limited to these two cases.Plasmids isolated from bacterial strains isolated in a hospitable mediummay possess the information for this type of resistance. For example,the plasmid JR66 (DATTA et HEDGES, J. Gen. Microbiol. (1973) 77:11)results in the resistance to bleomycins as well as to numerousaminosides for the cells of Escherichia coli which harbor it.

Another origin of genes which can result in a resistance phenotype tothe bleomycins is provided by the chromosomic DNA of Actinomycesproducing these antibiotics. Such a gene has been cloned from thechromosomic DNA of the strain of Streptomyces verticillius (SV) ATCC21890 producing phleomycins and resistance to at least 200 μg/ml of thephleomycins or of commercial bleomycine. The DNA of the strain 21890prepared according to the method of MARMUR (J. Mol. Biol. 1961, 3:208)is purified on DEAE-cellulose then partly digested with MboI to obtainfragments comprised between 2 and 6 kilobases. The product of theligation of these fragments with the DNA of pIJ702 opened by BglII isused to transform the protoplasts of Streptomyces violaceonigeraccording to the method described by THOMPSON et al. J. Bacteriol.(1982) 151:668. Arrest of the contacting of the protoplasts with the DNAin the presence of polyethylene glycol is done by dilution to 1/10 in ahypertonic medium and spreading at this dilution on dishes containingthe non-selective R2 regeneration medium. After a week of incubation,the surface of the dishes is scraped and the sporesmycelium suspensionis spread on dishes of GAPY medium supplemented with 20 μg/ml ofphleomycine and 50 μg/ml of Nosiheptide (antibiotic close tothiostrepton). In the course of three separate experiments, a singlestable clone, capable of developing in the presence of 20 μg ofphleomycin, was isolatable. The plasmid pUT212 extracted from this clonecan transform Streptomyces violaceoniger for the character of resistanceto the phleomycins but also to other antibiotics tested: commercialbleomycin, pepleomycin, bleomycin A5 and tallysomycin.

EXAMPLE X

The transposon Tn5 functional in a large number of Gram⁻ bacteria isvery much used in molecular biology for mutagenesis experiments byinsertion. The insertion of the transposon Tn5 in a chromosomic,plasmidic or phagic DNA, is followed by means of the character ofresistance to kanamycin. Resistance to bleomycin can just as well beexploited for the selection. This has been checked in particular withdifferent insertion mutants of the Tn5 in the chromosomic DNA inRhizobium meliloti strain 41. All the mutants examined are shown to beresistant at the same time to kanamycin and to bleomycins. It must bepointed out that the plasmid pRme:Tn5 used for these experiments resultsin a resistance higher than 300 μg/ml of commercial bleomycin for thecells of the strain 41 of Rhizobium meliloti whereas in the absence ofthe plasmid the cells are killed at a concentration equal or less than 5μg/ml in the medium TY at pH 8.

In the field of plant molecular biology the labels of resistance to thebleomycins can be shown to have great utility. Thus the cells derivedfrom protoplasts of tobacco are killed at 1 μg/ml of commercialbleomycine in the medium C (MULLER et al., Physiol. Plant. (1983)57:37). The gene of structure Tn5 Phleo^(r) (FIG. 2) placed under thedependance of promoter and terminal sequences specific to plant cellsshould be exploitable beneficially for gene transfer in plants. Aplasmid carrying the gene Tn5 Phleo^(r) under the control of thesequences 5' and 3' not translated of nopaline synthetase has beenconstructed. The DNA of the plasmid pLGV23 Neo (HERRERA-ESTRELLA et al.,EMBO J. (1983) 6:987) extracted from the strain of Escherichia coli GM99 (dam⁻) is linearized by BamHI and partly digested with BclI. Thefragment of approximate size 6.7 kilobases is electroeluted thenligatured with the small fragment BglII-BamHI of pUT6 to give theplasmid pUT400 (FIG. 9). The cells of HB101 carrying pUT400 are weaklyresistant to the bleomycins (c.m.i.:5 μg/ml of commercial bleomycin).Plasmid pUT400 should be shown to be particularly interesting for thecloning of genes in plants either by means of Agrobacterium tumefaciensor Agrobacterium rhizogenes and their respective plasmid Ti and Ri or bydirect transfection of protoplasts of plant cells after encapsidation ofthe DNA in liposomes (CABOCHE et al., Colloque Biologie MoleculaireVegetable, ORSAY (1984) Poster p. 62).

By way of reference for the identification of the antibiotics of thefamily of phleomycins, the "Handbook of Antibiotic Compounds" ed. BerdyCRC press, 1980, volume 4 could be consulted.

Pepleomycin is particularly described in the article of TAKAHASHY etal., J. Antibiotics 1979, 27, 36.

We claim:
 1. A method of transforming eukaryotic and prokaryotic cellssensitive to an antibiotic of the phleomycin family to confer resistanceagainst said antibiotic, wherein the eukaryotic and prokaryotic cellsare selected from the group consisting of Escherichia coli, Bacillussubtilis, mouse cells, Saccharomyces cerevisiae and Rhizobium meliloti,comprising:inserting a phleo^(r) gene of transposon Tn5, a phleo^(r)gene of plasmid pUB110, or a phleo^(r) SV gene of plasmid pUT212 intothe eukaryotic or prokaryotic cell to transform the cell to conferresistance against the antibiotic.